Browsing by Subject "function discovery"
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Item Discovery of Deaminase Activities in COG1816(2013-04-24) Goble, Alissa MImproved sequencing technologies have created an explosion of sequence information that is analyzed and proteins are annotated automatically. Annotations are made based on similarity scores to previously annotated sequences, so one misannotation is propagated throughout databases and the number of misannotated proteins grows with the number of sequenced genomes. A systematic approach to correctly identify the function of proteins in the amidohydrolase superfamily is described in this work using Clusters of Orthologous Groups of proteins as defined by NCBI. The focus of this work is COG1816, which contains proteins annotated, often incorrectly, as adenosine deaminase enzymes. Sequence similarity networks were used to evaluate the relationship between proteins. Proteins previously annotated as adenosine deaminases: Pa0148 (Pseudomonas aeruginosa PAO1), AAur_1117 (Arthrobacter aurescens TC1), Sgx9403e and Sgx9403g, were purified and their substrate profiles revealed that adenine and not adenosine was a substrate for these enzymes. All of these proteins will deaminate adenine with values of kcat/Km that exceed 105 M-1s-1. A small group of enzymes similar to Pa0148 was discovered to catalyze the hydrolysis of N-6-substituted adenine derivatives, several of which are cytokinins, a common type of plant hormone. Patl2390, from Pseudoalteromonas atlantica T6c, was shown to hydrolytically deaminate N-6-isopentenyladenine to hypoxanthine and isopentenylamine with a kcat/Km of 1.2 x 107 M^-1 s^-1. This enzyme does not catalyze the deamination of adenine or adenosine. Two small groups of proteins from COG1816 were found to have 6-aminodeoxyfutalosine as their true substrate. This function is shared with 2 small groups of proteins closely related to guanine and cytosine deaminase from COG0402. The deamination of 6-aminofutalosine is part of the alternative menaquinone biosynthetic pathway that involves the formation of futalosine. 6-Aminofutalosine is deaminated with a catalytic effeciency of 105 M-1s-1 or greater, Km?s of 0.9 to 6.0 ?M and kcat?s of 1.2 to 8.6 s-1. Another group of proteins was shown to deaminate cyclic- 3?, 5? -adenosine monophosphate (cAMP) to produce cyclic-3?, 5?-inosine monophosphate, but will not deaminate adenosine, adenine or adenosine monophosphate. This protein was cloned from a human pathogen, Leptospira interrogans. Deamination may function in regulating the signaling activities of cAMP.Item Functional Exploration and Characterization of the Deaminases of Cog0402(2014-02-24) Hitchcock, Daniel StephenHigh throughput sequencing technology and availability of this information has changed the way enzyme families can be studied. Sequence information from large public databases such as GenBank and UniProtKB can easily retrieved for the purpose of identifying unique enzymatic activities. The strategy adopted for this study is to identify characterized enzymes and the sequence features which give rise to their substrate specificity. Homologues of these enzymes are retrieved, and any active site variations can be readily identified. Cluster of Orthologous Groups (cog) 0402 is a family of enzymes which comprise a portion of the amidohydrolase superfamily. This group catalyzes a deamination reaction, releasing free ammonia and replacing it with a tautomerized oxygen. Cog0402 is most well known for guanine and cytosine deaminase, however other functions exist. One such function was that of S-adenosylhomocysteine deaminase, which was related to a large group of uncharacterized enzymes. These enzymes were predicted by us to deaminate 5?-modified adenosines. The enzymes were physically characterized these predictions were confirmed and a 5?-deoxyadenosine deaminase was discovered in addition to an 8-oxoadenine deaminase. During this study it was noted that background isoguanine deaminase activity was found at appreciable rates in E. coli. This activity was purified and identified using nanoLC-MS/MS and found to be caused by E. coli cytosine deaminase. E. coli cytosine deaminase itself is found in a cluster of uncharacterized enzymes with a single amino acid difference in the active site. Representative enzymes were purified and a 5-methylcytosine deaminase was discovered. This enzyme is capable of rescuing thymine auxotrophs in the presence of 5-methylcytosine, and will confer sensitivity to 5-fluorocytosine. Finally, an enzyme distantly related to cytosine deaminase was purified and found to be a unique pterin deaminase. It was most efficient for oxidized pterin rings and would accept a variety of substituents on the C6 positions. Futhermore, it was thought to catalyze the first step of an undescribed pterin degradation pathway.Item Mechanistic and Functional Characterization of Lactonases of COG3618 in the Amidohydrolase Superfamily(2014-05-06) Hobbs, Merlin EricThe postgenomic era of scientific research has yielded an inundation of gene and protein sequences which are available in public databases. This torrent of sequences, literally in the millions, has altered the strategies, methodologies, and approaches taken toward function discovery and annotation. At the forefront, are interdisciplinary approaches; such as, genomic enzymology. Genomic enzymology bridges aspects of classical enzymology, structural and functional genomics, and comparative genomics. These methods also take advantage of evolutionarily related proteins, which have relatively similar sequence similarity but, yield different functions. The amidohydrolase superfamily (AHS), contains proteins of similar chemistry and topology, which are related to one another through a common ancestral progenitor. The AHS has been organized into smaller groups based only on sequence similarity, which are known as clusters of orthologous groups (COG). There are currently 24 COGs within this superfamily. Cog3618 is comprised of primarily lactonase enzymes. The first identified member of cog3618 was LigI, which catalyzes the reversible hydrolysis of 2-pyrone-4,6-dicarboxylate. The chemical mechanism was elucidated and it was determined that this protein is the first member of the AHS that does not require a metal cofactor for catalysis. The mechanistic characteristics of LigI, along with genomic enzymology, were utilized to predicted and identify the functions of two other COG members. BmulJ_04915 and BVU_0220 were identified as L-fucono-1,5-lactonase and L-galactono-1,5-lactonase, respectively. NMR analysis revealed that these enzymes give preference to 6-member lactones, as opposed to their more stable counterparts. In addition, it is proposed that L-galactono-1,5-lactone participates in a novel L-galactose catabolic pathway, which appears to converge with D-galacturonate degradation through the intermediate of an adjacent dehydrogenase (BVU_0222). BVU_0222 was determined to oxidize L-galactonate to D-tagaturonate, which is a pathway intermediate for the degradation of D-glucuronate.Item Mechanistic Studies and Function Discovery of Mononuclear Amidohydrolase Enzymes(2011-02-22) Hall, Richard StuartThe amidohydrolase superfamily is a functionally diverse group of evolutionarily related proteins which utilize metal cofactors in the activation of a hydrolytic water molecule and in the stabilization of the resulting tetrahedral intermediate. Members of this superfamily have been described which use one or two divalent transition metals. These metal cofactors are located in either or both of two active-site metal binding centers which are labeled as the Ma and MB sites. The goal of this research was to elucidate the nature of the reactions catalyzed by Ma and MB mononuclear members of the amidohydrolase superfamily. This was approached through comprehensive mechanistic evaluations of two enzymes which utilized the different metal sites. Nacetyl- D-glucosamine-6-phosphate deacetylase from E. coli (NagA) and cytosine deaminase from E. coli (CDA) served as models for mononuclear amidohydrolase superfamily enzymes which have evolved to utilize a single B-metal and a single a-metal for hydrolysis, respectively. This research elucidated the different properties imparted by the distinct a and B active sites and the specific interactions utilized by the enzymes for substrate binding and catalysis. These studies led to the eventual proposal of detailed chemical mechanisms and the identification of rate determining steps. Knowledge of sequence-function relationships was applied toward the discovery of function for enzymes related to cytosine deaminase and guanine deaminase. The first group of enzymes investigated was proposed to catalyze the fourth step in riboflavin and coenzyme F420 biosynthesis in Achaea. Three putative deaminases; Mm0823 from Methanosarcina mazei, MmarC7_0625 from Methanococcus maripaludis C7 and Sso0398 from Sulfolobus solfataricus were cloned and expressed. These proteins proved to be intractably insoluble. A second set of enzymes, Pa0142 from Pseudomonas aeruginosa PA01 and SGX-9236e (with crystal structure PDB: 3HPA) were found to catalyze the novel deamination of 8-oxoguanine, a mutagenic product of DNA oxidation. 9236e was cloned from an unidentified environmental sample of the Sargasso Sea. The closest homolog (98% identical) is Bcep18194_A5267 from Burkholderia sp. 383. Additionally, it was discovered that the proteins SGX-9339a (with crystal structure PDB: 2PAJ) and SGX-9236b catalyzed the deamination of isoxanthopterin and pterin-6- carboxylate in a poorly characterized folate degradation pathway. These enzymes were also from unknown environmental samples of the Sargasso Sea. The closest homolog of 9339a (88% identical) is Bxe_A2016 from Burkholderia xenovorans LB400. The closest homolog of 9236b (95% identical) is Bphyt_7136 from Burkholderia phytofirmans PsJN.